The major objective of this proposal is to elucidate cell-matrix interactions important during development of the cornea stroma. Our hypothesis is that cell-matrix interactions mediate the morphological and physiological changes which occur at this time. We will characterize the expression of integrin and non-integrin receptors during four stages of development: (1) within the periocular mesenchyme prior to migration of corneal fibroblast precursors; (2) upon initiation of migration of corneal fibroblast precursors; (3) when differentiation begins, upon contact with the primary stroma; (4) when fully differentiated corneal fibroblasts are depositing the orderly matrix of the secondary stroma. Integrins are heterodimeric cell surface receptors which effect morphological and physiological changes upon binding to extracellular matrix. We hypothesize that the stationary phenotype and orderly matrix deposition characteristic of corneal fibroblasts will be correlated with a precise organization of integrin heterodimers. This will be analyzed using immunoblotting, immunoprecipitation, various immunolocalization techniques, and flow cytometry. We have documented spatial and temporal differences in matrix deposition during the initiation of migration and differentiation of cornea fibroblasts/precursors. We hypothesize that these changes will correlate with changes in integrin expression, which will be analyzed using the same techniques. Non-integrin receptors may also be important during corneal development. Next, we will characterize the expression of non-integrin matrix receptors: NG2, which binds type VI collagen, and the hyaluronate receptor. Type VI collagen and hyaluronate exhibit changes in distribution during corneal development. Thus, we hypothesize that receptors for both these ligands will be important at this time. It will then be determined what in vitro culture conditions maintain expression of matrix receptors. Use of freshly isolated cells will enable us to do short term functional essays, but we hypothesize that by adjusting culture conditions we will be able to maintain expression for longer term studies. Finally, functional studies will determine which receptor is actually binding a particular ligand. We hypothesize that cells from different developmental stages will exhibit differential attachment, spreading and migration which can be correlated with matrix receptor expression. Antibody inhibition assays will assess the role of various integrin subunits/heterodimers and non-integrins in these processes. Characterization of cell-matrix interactions during corneal development will provide information concerning the fundamental processes regulating cornea transparency as well as those involved in wound repair.